EP2636675B1 - Diphosphonate compounds and preparation method and use thereof - Google Patents

Diphosphonate compounds and preparation method and use thereof Download PDF

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Publication number
EP2636675B1
EP2636675B1 EP11837502.1A EP11837502A EP2636675B1 EP 2636675 B1 EP2636675 B1 EP 2636675B1 EP 11837502 A EP11837502 A EP 11837502A EP 2636675 B1 EP2636675 B1 EP 2636675B1
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compound
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added
substituted
alkyl
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EP2636675A4 (en
EP2636675A1 (en
Inventor
Mingqi Li
Han Wang
Qimin Deng
Zuoyong Cheng
Maoliang Li
Yonglong Zeng
Xue JIANG
Yuchun Duan
Sanping Zhu
Dezhong Wen
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Chengdu Yunke Pharmaceutical Co Ltd
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Chengdu Yunke Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
    • C07F9/6539—Five-membered rings
    • C07F9/6541—Five-membered rings condensed with carbocyclic rings or carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C07F9/4003—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4025—Esters of poly(thio)phosphonic acids
    • C07F9/4028—Esters of poly(thio)phosphonic acids containing no further substituents than -PO3H2 groups in free or esterified form
    • C07F9/4031—Acyclic unsaturated derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/5475—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and selenium with or without oxygen or sulfur as ring hetero atoms; having nitrogen and tellurium with or without oxygen or sulfur as ring hetero atoms

Definitions

  • the present invention relates to a new diphosphonate compound, and to a method to prepare the above new diphosphonate compound.
  • Bone metabolic diseases are disorders of bone formation, bone resorption and deposition of bone minerals caused by disturbance of normal bone metabolism from congenital or acquired factors. Bone metabolic diseases include osteoporosis, vitamin D deficiency, vitamin C deficiency, renal osteodystrophy etc. Among these bone metabolic diseases, osteoporosis is a common and frequent disease. Osteoporosis is a systematic and systemic bone disease characterized by brittleness increase of bone and high risk of fracture caused by bone loss, bone tissue microstructure destruction.
  • osteoporosis As living standard of people continuously improves, life span of people prolongs and aging society comes, osteoporosis has become a common and frequent disease that seriously threatens health of the middle and old aged; particularly for menopause women, the balance of bone formation and bone resorption is disturbed by variation in estrogen level, resulting in massive bone loss, increase of bone resorption and/or descent of bone formation, causing osteoporosis, and even worse, osteoporotic fracture.
  • China is not only the country having the largest amount of population, but also the country having the largest amount of osteoporosis patients. Based on the fifth nationwide census, the male incidence of osteoporosis is 14.6% and the female incidence of osteoporosis is 61.8% in the old-aged above 60 years old, with an overall incidence of 6.97% and totally 88.26 million of middle and old aged threatened by occurrence of osteoporosis. To Mid 21 Century. China will come into the peak period of the aged society, the population above 60 constitutes 27% of the total population, reaching up to 400 million. As a result, the research and development on the drugs to prevent and treat osteoporosis have a great sense in improving the health of people and increasing life quality and also have a great social value.
  • the drugs used for treating osteoporosis mainly include four classes: one is bone resorption inhibitors such as a variety of diphosphonate compounds. Isopropyl isoflavones, calcitonin, estrogen and selective estrogen receptor modulator; one is bone formation-accelerating drugs such as fluoride, parathyroid hormone, insulin-like growth factor, protein synthetic hormone; one is ossification-accelerating drugs such as calcium, vitamin D and its derivatives; one is drugs for inhibiting the activity of osteoclast and enhancing bone formation such as strontium ranelate; and other classes such as traditional Chinese medicine.
  • bone resorption inhibitors such as a variety of diphosphonate compounds. Isopropyl isoflavones, calcitonin, estrogen and selective estrogen receptor modulator
  • bone formation-accelerating drugs such as fluoride, parathyroid hormone, insulin-like growth factor, protein synthetic hormone
  • ossification-accelerating drugs such as calcium, vitamin D and its derivatives
  • Diphosphonate drugs i.e. pharmaceutically-acceptable salts
  • Diphosphonate drugs are synthetic analogue of natural pyrophosphate, having the basic structure shown in formula I: as shown in formula I, R', R" lateral chains in diphosphonate molecular structure may affect its bone affinity.
  • R' is H, OH or halogen and the like groups
  • R' has a smaller influence on the bone affinity of diphosphonate drugs
  • the structure of R" is a main factor that affects the bone affinity of diphosphonate drugs.
  • R is diphosphonate salt not comprising nitrogen, represented by Etidronate, clodronate, and clinically applied in the 70s last century; the second is that R" structure comprises amino group, having a stronger ability of inhibiting bone resorption than the diphosphonate drugs without nitrogen, represented by Panidronate, alendronate.
  • R" is diphosphonate salt comprising nitrogen containing heterocycle, having an even stronger ability of inhibiting bone resorption and an easier clinical application, represented by Zoledronate which has a bone affinity over 10000 times stronger than Etidronate.
  • Diphosphonate drugs comprising N (comprising-NH 2 or comprising N heterocycle) or not comprising N both treat osteoporosis through inhibiting the activity of osteoclast and have a weak effect on osteoblast proliferation.
  • commercially available diphosphonate drugs have a more and more stronger effect on inhibiting osteoclast but also increase the risk of fracture, and moreover it has not reported that diphosphonate drugs have an effect on osteoblast proliferation.
  • the object of the present invention is to overcome the deficiency that currently available diphosphonate compounds have strong effect on inhibiting the activity of osteoclast but a weaker effect on osteoblast proliferation, and to provide a new diphosphonate compound and a method for preparing the same.
  • the diphosphonate compound provided in the present invention is capable of bi-directionally regulating osteoblast and osteoclast.
  • R 1 is preferably H, OH.
  • R 1 may also be halogen, preferably F, Cl or Br.
  • R 2 is wherein Ar is substituted aryl, the substituent is C1 ⁇ C6 alkyl substituted in different sites, C1 ⁇ C6 optionally substituted alkyl, halogen, -CN, -NO 2 or -OH.
  • R 3 is preferably Se.
  • R 6 , R 7 , R 8 and R 9 are preferably hydrogen.
  • the present invention also provides two methods for preparing the diphosphonate compound having the above formula II.
  • Tetraisopropyl methylene diphosphonate and NaH are performed with the proton activation reaction of methylene for 1h ⁇ 4h under -5°C ⁇ 40°C, with the mole ratio of NaH and tetraisopropylmetlrylene diphosphonate being 1:1 ⁇ 3:1;
  • the compound A comprising substituent of bromine and nitro are added to the reaction liquid resulted from step (1) to perform alkylation reaction under 60°C ⁇ 150°C for 2h ⁇ 5h, the mole ratio of compound A and tetraisopropyl methylene diphosphonate is 1:1 ⁇ 3:1; after reaction organic solvent is added to the reaction liquid for extraction, the organic phase is collected and rotaiyly-dried, and compound I with the methylene substituted by the compound comprising nitro group is obtained;
  • 10% Pd/C catalyst is added to the alcohol solution of compound I obtained in step (2) and hydrogen gas is inlet, reacted under room temperature with 1 to 20 atmospheric pressure for 18h ⁇ 48h, with the weight ratio of compound I and 10% Pd/C catalyst being 1:10 ⁇ 1:5, the solution is filtered and evaporated dried to obtain compound II;
  • Alkaline solution is added to the compound II obtained in step (3) under the temperature of -20°C ⁇ 5°C, the pH is adjusted to 7 ⁇ 14, then the organic solvent of o- seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride is added, and reacted under room temperature for 3h ⁇ 8h with the mole ratio of compound II and o- seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride being 1:1 ⁇ 1:5, the reaction solution is filtered, the precipitate is collected and washed with organic solvent and dried under 60°C ⁇ 100°C for 4h ⁇ 24h to obtain compound V;
  • Alcohol and concentrated hydrochloric acid are added to the compound V obtained in step (4) with 1g compound V added 5mL ⁇ 50mL alcohol solution and 5mL ⁇ 60mL concentrated hydrochloric acid, refluxed under 90°C ⁇ 120°C for 4h ⁇ 10h.
  • the solvent is removed through reduced pressure distillation, the residue is washed with alcohol, and the solid is vacuum-dried under 50°C ⁇ 120°C for 4h ⁇ 10h to obtain the diphosphonate compound having formula II.
  • the organic solvent in step (2) may be methylene dichloride, methenyl chloride, ethyl acetate or petroleum ether.
  • the alkaline solution in step (4) is NaHCO 3 solution.
  • the organic solvent of o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloridein step (4) is ethylether, methylene dichloride, methenyl chloride or ethyl acetate.
  • the o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride in step (4) is prepared from the following method:
  • the alkaline solution of Se/S ion is obtained from the reaction of selenium powder or sulfur powder with potassium borohydride, and NaOH in water for 3h ⁇ 10h, wherein the mole ratio of potassium borohydride, NaOH and selenium powder/sulfur powder is 1:10 ⁇ 5:1;
  • the diazonium salt solution is obtained from the reaction of the hydrochloric acid solution of anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent with NaNO 2 solution under room temperature for -20°C ⁇ 5°C; the mole ratio of NaNO 2 and anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent is 1:1 ⁇ 3:1.
  • the alkaline solution of Se/S ion is obtained from the reaction of selenium powder or sulfur powder with potassium borohydride and NaOH in water for 3h ⁇ 10h, wherein the mole ratio of potassium borohydride, NaOH and selenium powder/sulfur powder is 1:10 ⁇ 5: 1;
  • the diazonium salt solution is obtained from the reaction of the hydrochloric acid solution of anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent (compound C) with NaNO 2 solution under -20°C ⁇ 5°C; the mole ratio of NaNO 2 and compound C is 1:1 ⁇ 3:1.
  • the diphosphonate compound having formula II with R1 being H may be prepared.
  • the above preparing method comprises the following steps:
  • the alkaline solution of Se/S ion is obtained from the reaction of selenium powder or sulfur powder with potassium borohydride and NaOH in water for 3h ⁇ 10h, wherein the mole ratio of potassium borohydride, NaOH and selenium powder/sulfur powder is 1:10 ⁇ 5:1:
  • the diazonium salt solution is obtained from the reaction of the hydrochloric acid solution of anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent (compound C) with NaNO 2 solution under -20°C ⁇ 5°C; the mole ratio of NaNO 2 and compound C is 1:1 ⁇ 3:1.
  • the diphosphonate compound having formula II with R1 being H may be prepared.
  • the above preparing method comprises the following steps:
  • Another method for preparing the above diphosphonate compound having formula II comprises the following steps:
  • the organic solvent in step (1) may be ethyl ether, methylene dichloride, methenyl chloride or ethyl acetate.
  • step (4) the method for preparing o- seleniumchloroylbenzoyl chloride or o-sulfachloroylbemoyl chloride in step (4) is described as above.
  • the diphosphonate compound having formula II with R1 being OH may be prepared.
  • the above preparing method comprises the following steps:
  • Existing nitrogen-containing diphosphonate are the most widely used drug for treating osteoporosis, such as commercially available Alendronate and Zoledronate, which mainly inhibit the farnesyl diphosphate synthase and therefore reduce the protein level in osteoclast, thus inhibiting the activity of osteoclast and treating osteoporosis.
  • the activity of existing nitrogen-containing diphosphonate and its salt becomes higher, i.e. the effect of these compounds on inhibiting osteoclast become stronger, the effect on osteoblast proliferation is weak, which may increase the risk of fracture.
  • the present invention provides a new diphosphonate compound and a method for preparing the same and an application of the same, and moreover the structural features of the compound are identified.
  • alendronate sodium is commercially available and widely used, is taken as positive control, and the results indicate that the new diphosphonate compound provided in the present invention exhibits an activity in inhibiting osteoclast equivalent to alendronate sodium, and a higher activity in affecting the proliferation of osteoplast than the positive control compounds, but the positive control exhibits a weaker effect on osteoplast proliferation.
  • an administration schedule for the diphosphonate compound of the present invention is provided.
  • R 4 and R 5 are independently selected from the group consisting of Hydrogen, halogen, -CN, -NO 2 , -OH, -OR', -COOR', -OCOOR', -COR', -CON(R') 2 , -OCON(R') 2 , -SR, -SO 2 R, -SO 2 N(R') 2 , -SOR' group, C1 ⁇ C10 alkyl, C1 ⁇ C10 substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl aryl or substituted aryl, wherein each of R and R'is independently selected from the group consisting of H, alkyl, aryl, substituted alkyl or substituted aryl; R 4 and R 5 may also constitute 3 ⁇ 7 carbons of cyclo alkane, the cyclo alkane may be substituted by C1 ⁇ C6 alkyl, C1 ⁇ C6 substituted alky
  • R1 in the exemplified diphosphonate compound is H, it may be prepared by the method below:
  • Tetraisopropyl methylene diphosphonate and NaH are performed with the proton activation reaction of methylene for 1h ⁇ 4h under -5°C ⁇ 40°C, with the mole ratio of NaH and tetraisopropyl methylene diphosphonate being 1:1 ⁇ 3:1;
  • the compound A substituted by bromine and nitro is added to the reaction liquid resulted from step (1) to perform alkylation reaction under 60°C ⁇ 150°C for 2h ⁇ 5h, the mole ratio of compound A and tetraisopropyl methylene diphosphonate is 1:1 ⁇ 3:1; after reaction organic solvent is added to the reaction liquid for extraction, the organic phase is collected and rotaiyly-dried, to obtain compound I with the methylene substituted by the compound comprising nitro group;
  • 10% Pd/C catalyst is added to the alcohol solution of compound I obtained in step (2) and hydrogen gas is inlet, reacted under room temperature with 1 to 20 atmospheric pressure for 18h ⁇ 48h with the weight ratio of compound I and 10% Pd/C catalyst being 1:0.5 ⁇ 1:5, and then filtered and evaporated dry to obtain compound II;
  • the alkaline solution of Se/S ion is reacted with diazonium salt solution under 60°C ⁇ 100°C for 2h ⁇ 8h, pH of the reaction liquid is adjusted with acid to less than 5, the precipitate is filtered, and the filtration residue is washed with water, alkaline solution is added to dissolve the precipitate, and then the solution is filtered, the filtrate is acidized to pH less than 5, the precipitate is obtained, collected and the precipitate is dried under 80°C ⁇ 150°C to obtain diselenum/disulfide compound (compound III);
  • Alcohol and concentrated hydrochloric acid are added to the compound V obtained in step (4), with 1g compound V added 5mL ⁇ 50mL alcohol solution and 5mL ⁇ 60mL concentrated hydrochloric acid, and refluxed under 90°C ⁇ 120°C for 4h ⁇ 10h.
  • the solvent is removed through reduced pressure distillation, and the residue is washed with alcohol, and the solid obtained is vacuum-dried under 50°C ⁇ 120°C for 4h ⁇ 10h, to obtain the diphosphonate compound having formula II.
  • the organic solvent in step (2) may be methylene dichloride, methenyl chloride, ethyl acetate or petroleum ether.
  • the alkaline solution in step (4) is NaHCO 3 solution
  • the organic solvent of o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride is ethylether, methylene dichloride, methenyl chloride, ethyl acetate.
  • step (4) the o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride in step (4) is prepared from the following method:
  • the alkaline solution of Se/S ion is obtained from the reaction of selenium powder or sulfur powder with potassium borohydride, and NaOH in water for 3h ⁇ 10h, wherein the mole ratio of potassium borohydride, NaOH and selenium powder/sulfur powder is 1:10 ⁇ 5: 1;
  • the diazonium salt solution is obtained from the reaction of the hydrochloric acid solution of anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent with NaNO 2 solution under room temperature for -20°C ⁇ 5°C; the mole ratio of NaNO 2 and anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent is 1:1-3:1.
  • the alkaline solution of Se/S ion is obtained from the reaction of selenium powder or sulfur powder with potassium borohydride and NaOH in water for 3h ⁇ 10h, wherein the mole ratio of potassium borohydride, NaOH and selenium powder/sulfur powder is 1:104 ⁇ 5:1;
  • the diazonium salt solution is obtained from the reaction of the hydrochloric acid solution of anthranilic acid containing substituent, anthranilate cyclohexane containing substituent or ⁇ -alanine containing substituent (compound C) with NaNO 2 solution under -20°C ⁇ 5°C; the mole ratio of NaNO 2 and compound C is 1:1 ⁇ 3:1.
  • R1 in the exemplified diphosphonate compound is OH, it may be prepared by the method below:
  • the organic solvent of o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride is added into pH 7 ⁇ 14 ⁇ -amino acid methylester (compound B), and reacted under room temperature for 3h ⁇ 8h, with the mole ratio of o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride and compound B being 1 ⁇ 3:1, the reaction liquid is filtered, and the precipitate is washed with ethylether, and dried under 50°C ⁇ 100°C for 4h ⁇ 24h, to obtain compound VII;
  • Alcohol and concentrated hydrochloric acid are added to the compound VII obtained in step (1), with 1g compound VII added 2mL ⁇ 50mL alcohol and 5mL ⁇ 50mL concentrated hydrochloric acid, and refluxed under 90°C ⁇ 120°C for 4h ⁇ 10h, the solvent is removed through reduced pressure distillation, the residue is recrystallized with alcohol, to obtain the compound VIII;
  • step (2) The compound VII obtained in step (2) is reacted with phosphorous acid and phosphorus trichloride under 90°C ⁇ 120°C for 2h ⁇ 6h, water is added into the reactant, with 1g compound VIII added 1mL ⁇ 50 mL water, and refluxed under 90°C ⁇ 110°C for 1h ⁇ 3h, with the mole ratio of compound VIII and phosphorous acid being 1: 1 ⁇ 1:5, and the mole ratio of compound VIII and phosphorus trichloride being 1:2 ⁇ 1:6, the reaction liquid is filtered, alcohol solution is added into the filtrate and put still under -5°C ⁇ 50°C for 12h ⁇ 48h, and suction-filtered, and the solid is washed with 5°C ⁇ 15°C cold water, and vacuum-dried under 50°C ⁇ 120°C for 4h ⁇ 10h, to obtain the diphosphonate compound (compound IX) having formula II.
  • the organic solvent in step (1) may be ethyl ether, methylene dichloride, methenyl chloride or ethyl acetate.
  • step (4) The method for preparing o-seleniumchloroylbenzoyl chloride or o-sulfachloroylbenzoyl chloride in step (4) is described as above.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-benzyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the white solid is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-benzyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-benzyl-metlrylene-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the white solid is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-benzyl-1-hydroxyl-methylenediphosphonic acid.
  • the diphosphonate compound in this embodiment is 2-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-ethyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the white solid is 2-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-ethyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 2-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-ethyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the white solid is 2-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-benzyl-1-hydroxyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(4,5-dimethyl--1,2-seleniumazolidine-3-ketone)-benzyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the light yellow solid is 4-(4,5-dimethyl--1,2-seleniumazolidine-3-ketone)-benzyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(4,5-dimethyl-1,2-seleniumazolidine-3-ketone)-benzyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the method for preparing the compound comprises the following steps:
  • the light yellow solid is 4-(4,5-dimethyl-1,2-seleniumazolidine-3-ketone)-benzyl-1-hydroxyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylcyclohexano[d][1,2]selenazole-2(3H))-benzyl-methylene-diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 5, but the only difference is that 2-amino cyclohexane carboxylic acid replaces 2-methyl- ⁇ butyrine to obtain white solid.
  • the white solid is 4-(3-carbonylcyclohexano[d][1,2]selenazole-2(3H))-benzyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3 -carbonylcyclohexano[d][1,2] selenazole-2(3H))-benzyl-1-hyclroxyl-methylenectiphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 6, but the only difference is that 2-methyl- ⁇ -butyrine is replaced by 2-aminocyclohexanecarboxylic acid to obtain white solid.
  • the white solid is 4-(3-carbonylcyclohexano[d][1,2]selenazole-2(3H))-benzyl-1-hydroxyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonyl-5-chlorobenzo[d][1,2]selenazole-2(3H))-benzyl-metlrylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 1, but the only difference is that anthranilic acid is replaced by 2-amino-5-chlorine-benzoic acid to obtain white-like solid 4-(3-carbonyl-5-chlorobenzo[d][1,2]selenazole-2(3H))-benzyl-methylene diphosphonic acid.
  • the white solid is 4-(3-carbonyl-5-cyclohexano[d][1,2]selenazole-2(3H))-benzyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonyl-5-chlorobenzo[d][1,2]selenazole-2(3H))-benzyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 2, but the only difference is that anthranilic acid is replaced by 2-amino-5-chlorine-benzoic acid to obtain white-like solid.
  • ESI-MS 512. Element analysis: Se 15.6%, P 12.0%.
  • the white solid is 4-(3-carbonyl-5-cyclohexano[d][1,2]selenazole-2(3H))-benzyl-1-hydroxyl-methylenediphosphoni c acid.
  • the diphosphonate compound in this embodiment is 4-(2-methylbenzo[d][1,2]selenazole-3(2H)-ketone)-benzyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 1, but the only difference is that 4-nitrobenzyl bromide is replaced by 4-nitromethylbenzyl bromide to obtain white solid.
  • 1H-NMR (DMSO, ⁇ ) 2.20 ⁇ 2.41 (t,1H); 3.05 ⁇ 3.19 (d,2H); 5.171 ⁇ 5.192 (S,2H); 7.316 ⁇ 7.334 (d,2H); 7.450 ⁇ 7.470 (t,1H); 7.512 ⁇ 7.530 (d,2H); 7.650 ⁇ 7.694 (t,1H); 7.879 ⁇ 7.896 (d,1H); 8.089 ⁇ 8.112 (d,1H); 10.321 ⁇ 10.507 (S,4H).
  • ESI-MS 476. Element analysis: Se 16.7%, P 13.1%.
  • the white solid is 4-(2-methylbenzo[d][1,2]selenazole-3(2H)-ketone)-benzyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(2-methylbenzo[d][1,2]selenazole-3(2H)-ketone)-benzyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 2, but the only difference is that methyl-(4-aminophenyl) acetate is replaced by methyl-a-methyl-p-amino phenyl acetate to obtain white solid.
  • the white solid is 4-(2-methylbenzo[d][1,2]selenazole-3(2H)-ketone)-benzyl-1-hydroxyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-phenylethyl-methylenediphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 1, but the only difference is that 4-nitrobenzyl bromide is replaced by 4-nitro-(2-bromomethyl)-benzene to obtain light yellow solid.
  • 1H-NMR (DMSO, ⁇ ) 1.35 ⁇ 1.49 (t,1H); 1.65 ⁇ 1.79 (m,2H); 3.00 ⁇ 3.12 (t,2H); 7.336 ⁇ 7.41 (d,2H); 7.453 ⁇ 7.475 (t,1H); 7.510 ⁇ 7.534 (d,2H); 7.651 ⁇ 7.693 (t,1H); 7.882 ⁇ 7.900 (d,1H); 8.088 ⁇ 8.115 (d,1H); 10.329 ⁇ 10.515 (S,4H).
  • ESI-MS 476. Element analysis: Se 16.6%, P 13.2%.
  • the white solid is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-phenylethyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-phenylethyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 2, but the only difference is that methyl 4-amino phenyl acetate is replaced by methyl 4-nitro-phenylpropionatere to obtain white solid.
  • ESI-MS 492. Element analysis: Se 16.2%, P 12.5%.
  • the white solid is 4-(3-carbonylbenzo[d] [1,2]selenazole-2(3H))-phenylethyl-1-hydroxyl-methylene diphosphonic acid
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]sulfonazole-2(3H))-benzyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 1, but the only difference is that selenium powder is replaced by sulfur powder to obtain yellow solid.
  • 1H-NM (DMSO, ⁇ ) 2.11 ⁇ 2.27 (t,1H); 2.98 ⁇ 3.12 (d,2H); 7.121 ⁇ 7.325 (d,2H); 7.352 ⁇ 7.371 (t,1H); 7.402 ⁇ 7.464 (d,2H); 7.501 ⁇ 7.592 (t,1H); 7.610 ⁇ 7.741 (d,1H); 7.918 ⁇ 8.002 (d,1H); 10.198 ⁇ 10.693 (S,4H).
  • ESI-MS 415. Element analysis: S 15.1%, P 14.9%.
  • the yellow solid is 4-(3-carbonylbenzo[d][1,2]sulfonazole-2(3H))-phenylethyl-methylene diphosphonic acid.
  • the diphosphonate compound in this embodiment is 4-(3-carbonylbenzo[d][1,2]sulfonazole-2(3H))-benzyl-1-hydroxyl-methylene diphosphonic acid, having the following formula:
  • the diphosphonic acid compound is prepared from the same synthesis process with embodiment 2, but the only difference is that selenium powder is replaced by sulfur powder to obtain yellow solid.
  • 1H-NMR (DMSO, ⁇ ) 3.07 ⁇ 3.21 (S,2H); 7.310 ⁇ 7.332 (d,2H); 7.350 ⁇ 7.369 (t,1H); 7.517 ⁇ 7.537 (d,2H); 7.562 ⁇ 7.593 (t,1H); 7.614 ⁇ 7.642 (d,1H); 7.801 ⁇ 7.872 (d,1H); 9.313 (S,1H); 10.409 (S,4H).
  • ESI-MS 431. Element analysis: S 14.6 %, P 14.3%.
  • yellow solid is 4-(3-carbonylbenzo[d][1,2]selenazole-2(3H))-phenylethyl-1-hydroxyl-methylene diphosphonic acid.
  • MG63 osteoblasts Key Laboratory of Ministry of Education for Biological Therapy of Human Diseases, SCU
  • cells adhere to wall and grow, and the endochylema begins to extend, fresh F-12 culture media containing 10% fetal bovine serum is added and replaced every 48 hours.
  • trypsin is used for digestion so that adherent cells become loose and round-shaped, and the digestion liquid is extracted out and added with fresh F-12 culture media containing 10% fetal bovine serum to terminate digestion.
  • Cells are pipetted from the culture flask, adjusted to the desired density, and transferred into the other culture flasks or culture plates.
  • MTT method is used to evaluate the effects of the drug on the activity of proliferation of osteoblasts MG63, wherein each well of 96-well plate is added with fresh MTT (5mg/mL) 100 ⁇ L, incubated under 37°C for 4h, and vibrated every a period of time. Then the supernatant is removed, each well is added with 200 ⁇ L DMSO (DMSO, Sigma corp.), vibrated on micro-oscillator for 10 min. Equivalent amount of liquid is transferred to an new plate and the absorbance value under 490nm is measured referring to table 1.
  • Table 1 indicates that, compared with the corresponding control groups (including blank and positive control) at the same time points, there is no statistical difference in absorbance value both at 24h and 48h (P>0.05) indicating the tested drugs and ALEN have no influence on proliferation of MG63 cells in the range of concentrations within 48h.
  • the alizarin red S(ARS) quantitative assay is applied, wherein each well of 6 well plate is added with 10 -5 mol/L, 10 -7 mol/L, 10 -9 mol/L of SC or ALEN, respectively, for 5d (1,25-(OH) 2 VitD 3 is added 48h before experiment termination for each well, and the final concentration of the vitamin is 10 -8 mol/L), and the culture media is extracted out. Then the cell was rinsed with PBS (pH7.2) gently for 3 times, fixed in 95% alcohol for 15min, and stained with 1% alizarin red S under room temperature for 30 min-45 min. Image Pro Plus 6.0 software (Media Cybernetics Inc.) is applied to measure the size of calcium nodules (orange red, diameter>200 ⁇ m).
  • each well is added with 1 mL extract (extract preparation: 800 mL 10% acetic acid with 200 mL anhydrous ethyl alcohol) extracted for 30min-45min, kept in dark, and shaken slightly.
  • the absorbance of 450nm wavelength is measured and recorded.
  • the concentration of alizarin red S in each well is calculated.
  • alizarin red S and calcium are linear correlated, the calcium deposition is evaluated according to the alizarin red S concentration.
  • the osteoclasts is cultured using a modified Chambers method, the new-born Japanese White Rabbit (1 week-age, provided by Experimental Animal Center of West China Medical Center of Sichuan University) is sacrificed by neck dislocation, and dipped in 75% ethanol for 5min, long bones are taken out under sterile condition, and soft tissue, periosteum as well as osteoepiphysis on the bone surface are removed in D-Hanks balanced salt solution, clean the bone shaft with ⁇ -MEM 2 times, and then cut off the long bone longitudinally in ⁇ -MEM full media (containing 15% fetal calf serum, penicillin 100 U/mL, streptomycin 100 ⁇ g/mL, 25m MHEPES, pH 7.0-7.2), scrape the medullar cavity of the bone gently until it is clean, and use a pipette (5mL syringe, 25G syringe needle) to rinse the medullar cavity repeatedly with culture media, and then the rinse solution is collected and filtered with cell
  • Results indicate that all concentrations of SC compounds and Alen, compared with blank, can inhibit the formation of osteoclasts and the inhibition is depending on the incubation time and concentrations of the tested drugs, but there is no significant difference between SC compounds and Alen indicating SC compounds and Alen have equivalent inhibition effect on the formation of osteoclasts. Generally, with the concentration and incubation time of the drugs increasing, TRAP + cell number obviously decreases.
  • the current in vitro study indicates that the bisphosphonates SC, on one hand, may inhibit differentiation and maturation of osteoclasts and reduce the metabolic activity of bone resorption; on the other hand, they may also considerably promote proliferation and differentiation of osteoblasts, enhance bone formation and regulate metabolic balance of bone formation/resorption, and finally restore the homeostasis of bone.
  • the in vitro study shows that the invention of SC compounds characteristic by their proliferation promotion effect to osteoblasts as well as the bisphosphonates' inhibition effect to osteoclasts. Therefore, the SC compounds of the present invention perform dual-directional regulation effect for osteoporosis treatment.

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